: Activation of the opioid receptor by agonists such as morphine is the primary biochemical mechanism by which opioids induced analgesia in humans. Prolonged stimulation of the opioid receptor by agonist can result in desensitization at the cellular and physiological level, resulting in the loss of analgesic efficacy. Receptor internalization and downregulation may be involved in the induction of tolerance. If these processes could be inhibited, it is possible that the development of tolerance to opioid agonists could be reduced or eliminated. This would be very beneficial to patients who experience chronic pain and require the pain relieving properties of analgesics in perpetuity. These same factors which influence the development of tolerance are also involved in reinforcing aspects that lead to addiction. Therefore, any agent which reduces internalization and downregulation of the opioid receptor will reduce the onset of pharmacological tolerance and also the addictive biochemical correlates associated with long-term treatment. This research proposal focuses on m- and d-opioid receptor phosphorylation under steady-state conditions and following agonist administration. Using mass spectrometry, we will determine the precise residues which are phosphorylated on the mu- and delta-opioid receptor in basal and various agonist-stimulated experimental paradigms. Human embryonic kidney cells expressing 6x-Histidine- and FLAG epitope-tagged mouse mu- or delta-opioid receptors will be used for these experiments. The 6xHistidine- and FLAG-epitopes will allow for immobilized metal ion affinity chromatography and immunoaffinity chromatography (IMAC). Purified proteins will be isolated via SDS/PAGE and extracted for mass spectrometric analysis. The use of mass spectrometry should allow for analytical determination of phosphorylated residues.